Hypercholesterolemia is a major factor in cardiovascular disease. In the USA, more than 35 million individuals have high total cholesterol and thus twice the normal risk of heart disease. The LDL receptor (LDLR) is critical to cholesterol regulation, as mutation of LDLR increases serum cholesterol levels and risk of cardiovascular disease. Increasing the level or activity of LDLR is an effective pharmacological mechanism employed by several cholesterol-lowering drugs now on the market. Statins, for example, inhibit HMG CoA reductase thus interfering with cholesterol synthesis, which upregulates hepatic LDLR activity and increases clearance of LDL from the bloodstream. Statins are widely prescribed, but are associated with adverse reactions in many patients;thus, new drugs of differing LDLR mechanism with reduced potential to cause side effects and/or potential for combination treatment employing reduced statin doses are currently being sought. An ubiquitin RING-finger E3 ligase known as Idol ( inducible degrader of the LDLR;also known as MYLIP and Mir), ubiquitylates LDLR on its cytoplasmic domain, resulting in its degradation. Idol is thus a promising new target for cholesterol-lowering drug discovery, as Idol inhibitors are expected to increase LDLR levels. Moreover, Idol inhibition provides a mechanistically distinct pathway to increase LDLR content/activity. The proposed project aims to discover small molecule inhibitors of Idol for lowering serum cholesterol by increasing LDLR levels and enhancing LDL clearance. Progenra has developed a novel assay that can detect the activity of virtually any E3 ligase;the assay has been used to find selective E3 ligase inhibitors, several of which are in pre-clinical development. Accordingly, the assay will be adapted and validated for high-throughput discovery of inhibitors of Idol, and a pilot screen will be conducted for inhibitors that are of potential utility as cholesterol- lowering therapeutic agents. The work will be divided into three Specific Aims. In the 1 year Phase I, the assay will be validated for inhibitors of both auto-ubiquitylation (Idol) and for inhibitors of ubiquitylation of the known substrate of human Idol. Cellular proof of concept will then be obtained experimentally using selected inhibitors from the screens. In Phase II, a more extensive screen and chemical optimization, ADME, and in vivo proof of concept and efficacy studies will be conducted, with the commercial goal of developing and marketing a pharmaceutical agent with single agent or combinatorial cholesterol lowering activity. PUBLIC HEALTH RELEVANCE: Elevated blood cholesterol is a major factor in cardiovascular disease;in the USA this condition affects more than 35 million individuals, who accordingly face increased risk of developing heart disease. The current drug of choice for lowering cholesterol is the family of statins, which work by increasing the binding of "bad" cholesterol to its receptors and thereby promote clearance of cholesterol from the bloodstream. Although widely prescribed, statins are associated with adverse reactions in many patients;thus, new drugs that would either work by a mechanism that leads to fewer or less severe side effects than those produced by the statins or be usable as part of a combination treatment regimen are currently being sought. Progenra has identified a promising new target for cholesterol-lowering drug discovery, an enzyme known as Idol, which conjugates ubiquitin to bad cholesterol receptors (LDLR), marking the receptor for degradation in the proteasome. Inhibitors of this target are expected to increase LDLR levels by preventing degradation of the receptor. Progenra will adapt and validate an assay for high throughput screening for inhibitors of Idol, screen its compound collection, and demonstrate that inhibitors from the screen affect LDLR in cells as predicted. In Phase II, selected inhibitors will undergo pre-clinical development, with the commercial goal of developing and marketing a pharmaceutical agent with single agent or combinatorial cholesterol lowering activity.